Project Summary/Abstract: The central nervous system (CNS) requires a tightly controlled environment free of various toxins and pathogens to provide the proper chemical composition for synaptic transmission. This environment is maintained by the `blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelial cells display specialized tight junctions and unusually low rates of transcellular vesicular transport (transcytosis). In concert with pericytes and astrocytes, this unique brain endothelial physiological barrier seals the CNS and controls substance influx and efflux. While BBB breakdown has recently been associated to initiation and perpetuation of various neurological disorders, an intact BBB is a major obstacle for drug delivery to the CNS. A limited understanding of the molecular mechanisms that control BBB formation has hampered our ability to manipulate the BBB in disease. Our recent discoveries changed our understanding of what makes the BBB impermeable. The BBB is formed by a single layer of endothelial cells that lines the walls of the brain's blood vessels. Historically, the restrictive feature of BBB has been attributed to the specialized tight junctions between adjacent endothelial cells. However, substances can also cross the endothelial layer by transcytosis, when material enters endocytic vesicles that are trafficked across the cell. We discovered that transcytosis is actively inhibited in brain endothelial cells to ensure BBB integrity. Our findings suggest that molecular pathways inhibiting transcytosis could be targeted to open the BBB for CNS therapeutics.We have also identified over 200 BBB candidate genes that are enriched in CNS endothelial cells compared to periphery endothelial cells. I propose to launch major new efforts leading to a major expansion in the scope of our work in the field of BBB. I will take the next eight years to bring my lab to the next level to (1) identify the full list of key BBB regulators in CNS endothelial cells, (2) understand what signals from non-endothelial cells maintain and regulate BBB permeability, and (3) determine how BBB permeability dynamically changes during different physiological and pathological conditions. We will also begin to work on translating findings from these studies to therapies. We will use a combination of mouse genetics, imaging, molecular, cell biology, and biochemical approaches. The experiments described here represent a major expansion in the scope of our work. Achieving the goals outlined here could have a major impact on neurology, enabling clinicians to open the BBB for transient delivery of drugs to the CNS, and conversely to close the BBB to slow the progression of neurodegenerative diseases. Given the transcriptome screens we have recently performed, the model systems we have devised, and the imaging tools we have recently developed, my lab is in a unique position to reveal the molecular and cellular mechanisms of the BBB.